PROJECT SUMMARY Accurate replication of the genome during cell proliferation is necessary for normal animal development and homeostasis. Disruption of the regulation or fidelity of replication contributes to many human pathologies, particularly cancer. Thus, a complete understanding of the mechanisms governing genome replication is paramount to human health. Replication of large genomes like that found in human cells requires the initiation of bi-directional DNA synthesis at thousands of individual locations on each chromosome. Executing this critical task requires a highly-regulated interaction between DNA and a large set of proteins whose activity must be coordinated with other cellular events such that all regions of the genome are replicated once and only once each cell division. Two decades of research by many laboratories has resulted in the identification of a set of 42 evolutionarily conserved polypeptides that are sufficient for initiation of DNA replication in a cell free setting, as well as how their activity is coordinated with the cell cycle. However, the mechanisms that determine how and where these factors interact with the genome in an intact animal cell, and how they are differentially activated to initiate DNA replication once they bind to the genome, are not well understood. These processes are modulated by the abundance of replication proteins, the chemical composition and relative compaction of chromatin, the folding of large domains of individual chromosomes, and the overall three-dimensional architecture of the genome within the nucleus. A major goal in the field is to determine how each of these levels of organization impact genome replication and stability in different cell types during development and in adult tissues. This project will specifically focus on how chromatin organization influences genome replication and stability during animal development. The basic building block of chromatin is the nucleosome, an octamer of histone proteins encompassed by ~147 base pairs of DNA. Each histone protein has an N-terminal tail that protrudes from the nucleosome core and is subject to a variety of chemical modifications (e.g. methylation, acetylation, and phosphorylation) that modulate chromatin organization and thus influence all aspects of genome function, including DNA replication. We have developed a method in Drosophila for engineering any desired histone tail mutation, providing us a means of manipulating chromatin organization that is not currently available in any other animal system. This genetic method will be combined with cell biological and next generation DNA sequencing methods to determine how chromatin organization modulates DNA replication in different cell types.